We build on the successful work in Phase II SBIR 5R43AI069631-02 in which a genetically attenuated Plasmodium falciparum (Pf) parasite (GAP) was produced and shown to generate aseptic Pf sporozoites (SPZ) that invade hepatocytes, but do not replicate. We will use this genetically attenuated double-mutant parasite deficient in the genes encoding SLARP and B9 (Pf?slarp?b9 GAP) to manufacture, characterize and release a corresponding PfSPZ Vaccine (PfSPZ (?slarp?b9) Vaccine, also known as PfSPZ-GA1 Vaccine) in compliance with current Good Manufacturing Practices (cGMPs), conduct pre-clinical Investigational New Drug application (IND)-enabling studies, develop a protocol for a phase 1 clinical trial, and submit an IND to the FDA. The work is based on a wealth of literature describing the robust protective immunity that can be induced in humans immunized with PfSPZ that invade hepatocytes, but arrest during liver stage development. Sanaria's PfSPZ Vaccine, consisting of radiation attenuated, aseptic, purified, cryopreserved PfSPZ, protected 6/6 (100%) of volunteers who received the highest dose of PfSPZ administered in the recent phase 1 clinical trial. The PfSPZ Vaccine is now being further assessed in clinical trials in the U.S. and Mali with additional trials pending soon in Tanzania, Germany, and Equatorial Guinea, and is on an accelerated development program leading to licensure in ~4 years for elimination campaigns in Africa and prevention of malaria in travelers. However, there would be manufacturing advantages and potential potency and regulatory advantages leading to significant cost of goods (COG) savings if the radiation-attenuated parasites were replaced with GAPs. We have demonstrated that eliminating the slarp and b9 genes leads to attenuation similar to radiation, and that in rodent malarias ?slarp?b9 SPZs elicit excellent protective immunity against SPZ challenge and do not lead to blood stage infection. All prior Pf GAPs showed leaky attenuation and breakthrough liver stage development in vivo or in vitro. To overcome this critical problem, our previous Phase II SBIR involved Pf strain engineering, phenotypic characterization, and proof-of-concept production of PfSPZ bearing knockout (KO) lesions in two attenuating genes (double KO strains) that would express a tight attenuated phenotype with no breakthroughs, and yet possess robust characteristics suited for our manufacturing process. A number of knock out parasites were produced and assessed, but only the slarp and b9 deficient clone of Pf met all of our requirements for moving forward in development. PfSPZ produced from the slarp and b9 deficient clone of Pf were completely attenuated at the early liver stage with no breakthroughs. A Master Cell Bank of this clone was made and an engineering production run was performed to demonstrate that the Pf?slarp?b9 GAP was suitable for producing aseptic, purified, cryopreserved PfSPZ. This was successfully accomplished. We now propose to initiate full pre-clinical development and IND submission in preparation for a phase 1 clinical trial of PfSPZ(?slarp?b9) Vaccine.